1

Antibodies to West Nile Virus and related Flaviviruses in wild boar, red foxes and 1

others mesomammals from Spain 2 3

Ana-Valeria Gutiérrez-Guzmána, Joaquín Vicente

a, Raquel Sobrino

a, Elisa Perez-Ramírez

b, 4

Francisco Llorenteb and Ursula Höfle

a* 5

6

aInstituto de Investigación en Recursos Cinegéticos IREC, (CSIC-UCLM-JCCM), Ciudad Real, Spain 7

bCentro de Investigación en Sanidad Animal del Instituto Nacional de Investigación y Tecnología Agraria y 8

Alimentaria (CISA-INIA), Ctra Algete-El Casar, s/n, 28130, Valdeolmos (Madrid), Spain 9

10

* Corresponding author: Instituto de Investigación en Recursos Cinegéticos IREC, (CSIC-UCLM-JCCM), 11

Ronda de Toledo s/n, 13005 Ciudad Real, Spain. Tel: +34 926 295450; fax: +34 926 295451. 12

E-mail addresses: ursula.hofle@uclm.es (U. Höfle). 13

14

15

Abstract 16

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) 17

that are raised extensively outdoors, as well as other wild mesomammals from south 18

central Spain and wild boar from Doñana National Park (DNP), were tested for antibodies 19

against West Nile Virus (WNV) and related flaviviruses by ELISA and against WNV by 20

VNT. Mean flavivirus seroprevalence according to ELISA was 20.4 ±7.8% (21 out of 103) 21

in red foxes, 12.6 ± 2.8% (69 out of 545) in wild boars, and 3.3 ± 2.7% (6 out of 177) in 22

Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence 23

in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the 24

serum samples limited interpretation of VNT to 28 samples, confirming WNV 25

seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, 26

microVNT negative samples suggest presence of non-neutralizing antibodies against WNV 27

or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands 28

2

for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar 29

and red foxes was not associated to wetland habitats. This is the first report of exposure of 30

red foxes to WNV. With view to use of the tested species as sentinels for flavivirus 31

activity, limited exposure of Iberian pigs that would be available for regular sampling, low 32

numbers of foxes collected and concentration of wild boar harvest in the winter season are 33

mayor drawbacks. 34

35

Keywords: Flavivirus, West Nile virus, Red fox, Wild boar, Iberian pig, Seroprevalence 36

37

1. Introduction 38

39 Flaviviruses have a worldwide distribution, and a number of them such as West Nile Virus 40

(WNV) are the cause of zoonoses of considerable importance for public health. The 41

flavivirus (family Flaviviridae) members of the Japanese encephalitis antigenic complex 42

(JEV) are maintained in a mosquito vector and bird reservoir cycle where humans, wild 43

and domestic mammals can be implicated as incidental hosts (Weissenböck et al., 2010). 44

Flavivirus activity has been rather limited in Spain until the last decade in which evidence 45

of circulation of different mosquito-borne flaviviruses has been found with increasing 46

frequency. In Spain, flavivirus activity main comprises three mosquito-borne flaviviruses 47

(WNV, USUV and BAGV; Figuerola et al., 2007; Busquets et al., 2008; Vazquez et al., 48

2010, 2011; Agüero et al., 2011). 49

WNV is a re-emerging zoonotic virus responsible of outbreaks in humans, domestic 50

animals (horses) and wildlife. In Spain, evidence of exposure to WNV has been found in 51

humans (Kaptoul et al., 2007; Anonymous, 2010), birds (Figuerola et al., 2007; Höfle et 52

al., 2008; López et al., 2008; Jiménez-Clavero et al., 2008), mosquitos (Vázquez et al., 53

2010, Sotelo et al, 2011a), and horses (Jiménez-Clavero et al., 2010; OIE, 2010). 54

3

USUV has caused disease and mortality in birds in Europe and recent human cases in Italy 55

(Pecorari et al., 2009), but as yet in Spain it has only been detected in mosquitoes 56

(Busquets et al., 2008; Vázquez et al., 2011) and no related disease has been reported in 57

birds or humans. In contrast, Bagaza virus (BAGV), a Flavivirus of the Ntaya serocomplex 58

that had never been detected before in Europe, was recently isolated from an outbreak of 59

lethal disease in free-living game birds in Southern Spain (Agüero et al., 2011). 60

Mammals can be naturally exposed to flaviviruses, either by bite from infected vectors, or 61

by ingestion of infected carrion or diseased prey (Austgen et al., 2004; Marra et al., 2004). 62

In fact, serological evidence of exposure to flaviviruses, mainly WNV, has been reported 63

in wild and domestic mammals from America, Africa, Asia and Eastern Europe (Root et 64

al., 2005; Bentler et al., 2007; Halouzka et al., 2008; Ohno et al., 2009, El-Harrak et al., 65

2011). In Spain, a report from 1980 described the presence of antibodies to flaviviruses in 66

rodents (Chastel et al., 1980). WNV seropositivity has been evidenced in horses (Jiménez-67

Clavero et al., 2010) and also clinical disease and mortality have been reported in this 68

species (OIE, 2010). Recently, we reported on exposure of wild juvenile ungulates, namely 69

wild boar and Iberian red deer (Cervus elaphus) to flaviviruses (Boadella et al., 2011). 70

Flavivirus exposure of free-living mammals with a broad geographic range and high 71

population density, such as wild boar (Acevedo et al., 2007), red fox (Vulves vulpes), and 72

other species could be a useful indicator of viral circulation and expand the knowledge on 73

virus ecology in Mediterranean ecosystems (Platt et al., 2008). Likewise, the extensively 74

reared Iberian pig could come in contact with vectors and thus be a useful source of 75

information on flavivirus activity in the region. 76

The objective of this work is to determine, through antibody detection, the degree of 77

exposure to flaviviruses (especially WNV) of wild boar, red foxes, and other medium sized 78

wild and domestic mammals in South-Central Spain and to further assess whether this 79

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information can be useful to monitor flavivirus activity in a given area. Also, the data 80

collected in this study from wild boar will be analyzed to test whether it is correct to 81

assume that the risk of exposure to flaviviruses is higher in wetland habitats, where a high 82

mosquito abundance (Dale and Knight, 2008) and a high concentration of a wide range of 83

wild bird species, either resident or migratory (López et al., 2008) is present, and during 84

specific seasons, in which mosquito activity is more likely. Thus, in this work we compare 85

antibody prevalence data among different geographical regions, habitats and seasons. The 86

effect of host factors such as age, sex and body condition on seroprevalence is also 87

assessed. 88

89

2. Material and methods 90

91 2.1 Study area 92

93

The study includes 23 sampling sites in south-western Spain (41º15 N and 38º04 N, 5º20 O 94

and 0º59 W, minimum altitude=244m, maximum altitude=2274m, relative humidity=64%, 95

mean temperature=14ºC), and one in Doñana National Park (DNP; 36º56 N, 6º21 W, mean 96

altitude=12m, relative humidity=80%, mean temperature=19ºC), in the Guadalquivir river 97

marshes in southern Spain (Figure 1). Of the 23 sampling sites in south-western Spain, 2 98

correspond to wetlands with abundance of migratory and resident birds; 9 are devoted to 99

agriculture and 12 are made up of typical Mediterranean forest in small mountain chains. 100

DNP is one of the main wetlands in Western Europe with high density and diversity of 101

resident and migratory birds. 102

103

2.2 Sampling 104

105

Blood samples of wild boar (n=545), red fox (n=103) and other wild mammals (European 106

wild cat Felis silvestris silvestris n=1, stone marten Martes foina n=6, European otter Lutra 107

5

lutra n=2, European badger Meles meles n=7, common genet Genetta genetta n=2) were 108

obtained from hunting drives, trapping programs for National Park management or from 109

animals found dead, mostly road-kills. Blood was collected from the thoracic cavity of 110

freshly dead animals. Blood of Iberian pigs (n=177) was collected from the infraorbital 111

sinus during the annual official active sanitary surveillance procedures (Real Decreto (RD) 112

1186/2006, published October 13th, 2006, now replaced by RD 599/2011, published April 113

29th 2011).Wild boars were sampled in 2007-2010, Iberian pigs in 2009-2010, red foxes in 114

2006-2008 and other small mammals in 2003-2007. Wild boar and red fox samples were 115

grouped by season (spring, summer, autumn, winter) and habitat type (wetland, 116

Mediterranean forest, and agricultural crops) for statistical analysis. 117

The age of wild boars was determined using dentition patterns, classifying them into 118

piglets (< 7 months), weaners (7 to 12 months), juveniles (12 to 24 months) and adults 119

(>2years; Matschke, 1967). When kidney data was available, the kidney fat index (KFI) 120

was obtained as indicator of body condition (Batcheler and Clarke, 1970). 121

Red fox samples were classified according to sex and age (juveniles and adults), and the 122

mean KFI was obtained when possible. 123

All Iberian pigs sampled were adults and body condition information was not available. 124

Upon arrival at the laboratory, blood samples were centrifuged for at least 10 min at 125

2,000g for serum separation and the serum was stored at -20º C until testing. 126

127

2.3 Serological tests 128

Presence of antibodies against WNV and closely related flaviviruses was analysed using a 129

commercial competitive enzyme–linked–immunosorbent assay (cELISA; ID Screen©® 130

West Nile Competition, ID Vet, Montpellier, France) based on purified whole WNV 131

antigen for detection of antibodies directed against the PrM-E envelope protein common to 132

flaviviruses. The test was performed according to manufacturer’s instructions. 133

6

To confirm ELISA positive samples, neutralizing antibody titers to WNV were determined 134

by a micro virus-neutralization test (micro VNT) previously described by Figuerola et al. 135

(2007), using Vero cells and WNV strain E101. 136

137

2.4 Molecular tests 138

To determine WNV and flavivirus genome presence, nucleic acids of available tissue 139

(spleen) of antibody-positive wild boar (n=69) were extracted (High Pure RNA Tissue Kit, 140

Roche Diagnostics, Barcelona, Spain), and analysed by real time reverse transcription-141

polymerase chain reaction (RRT- PCR) for WNV (TaqMan MGB PCR, QuantiTEC 142

Probe® RT-PCR, Qiagen, Madrid, Spain; Jiménez-Clavero et al., 2006), and Flavivirus 143

(QuantiTEC® SYBR®Green RT-PCR, Qiagen, Madrid, Spain) detection (Moureau et al., 144

2007). 145

146

2.5 Statistical analysis 147

A Chi square (χ2) test for homogeneity was used to compare the mean flavivirus 148

seroprevalence in wild boar between sampling sites and between DNP and the mean 149

seroprevalence from the combined sampling sites from south-western Spain. 150

To study factors that affect exposure to flaviviruses in wild boar we performed a 151

generalized mixed model (GzMM) where flavivirus seropositivity was the response 152

variable and “sex”, “age class”, “season”, “sampling year” and “habitat” were the 153

categorical explanatory variables. Sample origin was included a random variable. For this 154

analysis we used a binomial error and a logit link. Also a generalized mixed model 155

(GzMM) was performed in order to study the relationship between body condition (as 156

log10-transformed KFI, continuous response variable) and WNV antibody presence in 157

wild boar. We included “WNV seropositivity” (as categorical 0=absence, 1=presence), 158

7

“sex”, “age class”, “season” and “origin” (all of them as categorical) as explanatory 159

variables. “WNV seropositivity” interactions with “sex” and “age” were added to the 160

models. Sampling year was included as random variable. Identity error and a normal link 161

were applied. Finally the Chi square (χ2) test for homogeneity was used to compare 162

flavivirus seroprevalence in red foxes among age groups and according to sex. All analyses 163

were performed with the SPSS software package, version 19.0 (IBM SPSS Statistics, New 164

York, NY, USA). 165

166

3. Results 167

168 Antibodies against flaviviruses were detected by ELISA in 20.4 ±7.8% (21 out of 103) of 169

the red foxes, 12.6 ± 2.8% (69 out of 545) of the wild boars, in 3.3 ± 2.7% (6 out of 177) 170

of the Iberian pigs and in one stone marten (Table 1, Figure 1). In wild boar, a significantly 171

higher mean seroprevalence was found in DNP (27±7.1%) as compared to sampling sites 172

in south-western Spain (Figure 1, χ2 = 45.764, d.f. 6, p< 0.001) and the general mean 173

prevalence in south-western Spain (6.9±2.5%) respectively χ2=31.7, d.f. 1, p < 0.05). 174

Habitat, season and sampling year did not affect flavivirus seroprevalence in wild boar. 175

Also, flavivirus seroprevalence in wild boars and red foxes was apparently not affected by 176

sex and body condition. However, adult wild boar had a significantly higher flavivirus 177

antibody prevalence (19.7± 5.8%, 35 out of 178), than juveniles (7.8± 5.2%, 8 out of 103), 178

weaners (6.5± 4.7%, 7 out of 107), and piglets (5± 9.6%, 1 out of 20) (GLzMM, F=4.136, 179

d.f. 4, p<0,05, Figure 2). 180

Due to the strong haemolysis in sera from wild boars and red foxes, only 32% (21 wild 181

boar, 6 Iberian pig and 1 red fox samples) of the total samples tested by microVNT gave a 182

readable result. In wild boar, WNV neutralizing antibodies were found in 9 of the 69 183

ELISA positive samples (13%). More precisely 5 of 41 ELISA positive samples from DNP 184

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(12.2%), 2 of 7 ELISA positive samples from wetland habitats in south-western Spain 185

(28.6%), and 2 of 7 samples (28.6%) from Mediterranean forest habitat had WNV 186

neutralizing antibodies (Table 1, Figure 1). In the case of the Iberian pig, none of the 187

samples was haemolytic, and WNV neutralizing antibodies were detected in four (66.6%) 188

of the six ELISA positive samples (Figure 1). The stone marten ELISA positive sample 189

was negative by VNT. One sample of a red fox from a Mediterranean forest habitat 190

presented a high titre of WNV neutralizing antibodies, while neutralization titres for WNV 191

in wild boar and Iberian pigs were relatively low (Table 2). 192

All spleen samples analyzed by RRT-PCR were negative for the presence of WNV and 193

flavivirus genome. 194

195

4. Discussion 196

The results of the present study newly confirm the exposure of red foxes, Iberian pigs and 197

a stone marten from south-central Spain to flaviviruses. Mesomammals have previously 198

been confirmed to be exposed to WNV and other flaviviruses. However, to our knowledge, 199

this is the first report of flavivirus exposure in red fox and stone marten. Antibodies to 200

flaviviruses, mainly WNV, have been found in other members of the Canidae family, such 201

as gray foxes (Urocyon cinereoargenteus) or coyotes (Canis latrans; Bischof and Rogers, 202

2005; Bentler et al., 2007), and other mesomammals from North America (Dietrich et al., 203

2005; Root et al., 2005; Bentler et al., 2007; Gómez et al., 2008; Blitvich et al., 2009), but 204

had not been reported from Europe. Disease due to WNV has been documented in both 205

wolves (Canis lupus) and domestic dogs in connection with WN fever outbreaks in horses 206

and humans (Lanthier et al., 2004) and dogs have actually been proposed as potential 207

sentinels for WNV surveillance (Resnick et al., 2008). 208

9

The high flavivirus antibody prevalence rate evidenced in red foxes could be due to higher 209

exposure to frequent infected mosquito bites or to consumption of infected prey, although 210

this route of infection has only been reported in experimentally infected domestic cats and 211

naturally infected raptorial birds (Garmendia et al., 2000; Austgen et al., 2004). 212

Iberian pigs that could be more easily accessible than wild boar and red foxes for sampling 213

as sentinels were apparently less exposed to flavivirus. In a study including juvenile 214

individuals we showed that wild boar could be an interesting sentinel species for 215

flaviviruses surveillance (Boadella et al., 2011). As the frequently flavivirus positive red 216

foxes are generally available only in low numbers and as wild boar is mostly harvested in 217

winter, Iberian pigs that are farmed extensively and thus exposed to mosquitoes could be a 218

valuable alternative, not the least because they are accessible for sampling around the year, 219

and as blood samples are of better quality, than samples obtained from wild boar carcasses. 220

Our study shows however that exposure to flaviviruses in this species is much lower than 221

in wild boar in the same period. Previously, antibodies against WNV have been found in 222

feral swine in North America by ELISA with a mean prevalence in 2001-2004 (22.5%) 223

similar to the one encountered in DNP, and by VNT in 6.5% wild boar in the Czech 224

Republic (Gibbs et al., 2006; Halouzka et al., 2008). 225

In this study results for flavivirus seroprevalence in mesomammals by ELISA and WNV 226

seroprevalence by VNT differed. Currently, VNT is considered the gold standard to 227

confirm exposure to WNV (Dauphin and Zientara, 2006). Here, the high degree of 228

haemolysis present in the serum samples of wild boar and red foxes has only allowed 229

correct interpretation in a reduced number of samples (n=28). ELISA, on the other hand, is 230

less prone to haemolysis interference, but detects antibodies of cross related flaviviruses, 231

particularly viruses of the Japanese encephalitis serocomplex. Thus, in this study we 232

discuss flavivirus seroprevalence in general, as ELISA positive, microVNT negative 233

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samples could contain either non-neutralizing antibodies against WNV or antibodies to 234

other antigenically related flaviviruses. However, to date only the mosquito borne USUV 235

and BAGV, and the tick borne Spanish sheep encephalomyelitis virus (SSEV) have been 236

reported in the country (Marin et al., 1995; Busquets et al., 2008; Agüero et al., 2011). 237

The substantially higher flavivirus seroprevalence found in DNP as compared to south-238

central Spain (CLM) suggests a potential link to a habitat that favours vector abundance 239

and reservoir host (wild bird) presence. Nevertheless, no association of flavivirus 240

seroprevalence to wetland habitats could be established in wild boar in south-central Spain. 241

Flavivirus, namely WNV activity has been shown to vary with time due to climatic factors 242

that affect vector abundance (e.g. Platonov et al., 2008). In this study, flavivirus exposure 243

was detected between 2005 and 2010. For the years 2003 and 2004 only data for three 244

badgers, two genets, a wild cat and a stone marten were available, which is insufficient to 245

conclude about flavivirus activity in the study area. We were unable to detect a relation of 246

seroprevalence to year or season, however we also do not know about the duration of 247

antibody persistence in our test species, and most of our samples were from the winter (low 248

mosquito density) season which is when hunting drives take place. 249

The significantly higher seroprevalence found in adult wild boars in comparison to 250

juveniles, weaners and piglets (Figure 2), coincides with previous results in North 251

American feral swine (Gibbs et al., 2006) and in camels (El Harrak et al., 2011), and could 252

be explained by a longer time span of possible exposure or due to antibody persistence. In 253

adult pigs, antibodies to JEV have been found to persist for more than three years possibly 254

due to frequent re-inoculation by mosquito bites (Geevarghese et al., 1994), while other 255

experimental studies detected persistent antibodies in absence of re-infections only until 28 256

days post infection (Blitvich et al., 2003; Teehee et al., 2005). 257

11

Finally, in this work, we found antibodies against WNV and/or other flaviviruses but viral 258

genome was not detected, suggesting absence of active infection or low possibilities of 259

viral genome detection due to transitory viraemia or other host characteristics. Material 260

other than spleen that might have been more suitable for flavivirus genome detection was 261

not available for this study. 262

The results obtained in this study document the exposure of widely distributed wild and 263

domestic mammals in Spain to flaviviruses, specifically in areas where flavivirus activity 264

has been previously reported, but do not reveal Iberian pigs as good sentinel species for 265

flavivirus surveillance. With the samples available for this study we could neither 266

demonstrate increased exposure to flavivirus in wetlands nor a relation of flavivirus 267

exposure to season and thus mosquito abundance. The association of Flavivirus prevalence 268

with age suggests that juvenile individuals may be of more interest for surveillance. 269

Additional studies aimed at evaluating flavivirus circulation in other regions and the degree 270

of exposure of other widely distributed mammals would be of interest. 271

272

Conflict of interest statement 273

The authors have no conflict of interest. 274

275

Acknowledgements 276

We acknowledge the help and excellent comments that greatly improved the manuscript by 277

M.A. Jiménez-Clavero, and the inestimable help with sample collection of fellow students 278

in hunting drives and of the veterinary officers of the JCCM at Iberian pig farms. We are 279

also thankful to P. Acevedo for his help with Figure 1. This study has been supported by 280

projects PAC08-0296-7771 (JCCM), and AG2008-02504GAN. A.V. Gutierrez –Guzman 281

is a JCCM fellow (PAC08-029). 282

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283

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419

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422 423 424 425 426 427 428 429

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430 Table 1. Sampling effort by year and habitat type, and flavivirus and WNV seroprevalence by blocking 431

ELISA and WNV neutralization test in wild boar, red fox, and Iberian pigs. 432

2006 2007 2008 2009 2010

Species

Habitat

Wet-

land

locat

ion

n

ELIS

A pos.

VN

T

rd/+

n ELIS

A pos.

VN

T

rd/+

n ELIS

A pos.

VN

T

rd/+

n ELIS

A pos.

VN

T

rd/+

n ELIS

A pos.

VN

T

rd/

+

Mediterranean

forest

30 3 0/0 34 3 0/0 48 1 1/1 19 0 0/0

Wetland DNP 46 13 3/3 40 12 4/0 40 6 3/1 26 10 4/1

SW 30 1 1/1 55 2 2/2 76 2 2/0 2 0 0/0

Wild

boar

Agriculture

------

18 0 0/0 62 10 1/0 15 1 0/0 4 0 0/0

Total wild boar ----- 124 17 4/4 191 32 7/2 179 10 6/2 51 10 4/1

Mediterranean

forest

11 0 0/0 17 7 1/1 45 10 0/0

Wetland 11 1 0/0 -- -- -- -- -- --

Red fox

Agricultre 1 0 0/0 16 3 0/0 2 0 0/0

---- ----

Total red fox 23 1 0/0 33 10 1/1 47 10 0/0 --- ---

Mediterranean

forest

-- -- -- 20 0 --

Wetland -- -- -- -- -- --

Iberian

pig

Agriculture

---

----

---

86 3 3/3 71 3 3/1

Total Iberian pig --- --- --- 86 3 3/3 91 3 3/1

Total samples 23 1 0/0 157 27 5/5 238 42 7/2 265 13 9/5 142 13 7/2

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462

463

19

Table 2. Summary of antibody titres against WNV detected in free-living wild 464

boars, red foxes and Iberian pigs 465

466

Species 5 10 20 40 80 160 240 480

Wild boar 1 3 2 1 0 2 -- --

Red fox -- -- -- -- 1 -- -- 1

Iberian pig -- -- 1 1 1 1 -- --

467 468

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513

20

Figure 1. Flaviviruses seroprevalence in wild boar, red fox and Iberian pig from south-514

central, Spain. Stars indicate locations with VNT positive samples. 515

516

21

517

Figure 2. WNV/Flavivirus seroprevalence in wild boars of south central Spain increases 518

with age. 519

520

521 522